Lindbladh C, Persson M, Bülow L, Mosbach K
Department of Pure and Applied Biochemistry, University of Lund, Sweden.
Eur J Biochem. 1992 Feb 15;204(1):241-7. doi: 10.1111/j.1432-1033.1992.tb16630.x.
The two structural genes encoding galactose dehydrogenase (Pseudomonas fluorescens) and the beta subunit of luciferase (Vibrio harveyi) were fused in-frame in order to prepare and subsequently characterize an artificial bifunctional enzyme complex. This hybrid enzyme exhibited both galactose dehydrogenase activity and bioluminescence when expressed in Escherichia coli together with the alpha subunit of luciferase. The purified conjugate was used to study possible proximity effects in a sequential three-enzyme reaction with the bifunctional enzyme catalyzing the first and the last reaction. The intermediate enzyme, diaphorase, was added separately. The engineered enzyme system, comprising the galactose dehydrogenase/luciferase conjugate, could display a twofold higher bioluminescence in the overall enzyme reaction compared to a corresponding reference system with separate native enzymes. The increased bioluminescence obtained for the engineered enzyme system is proposed to be due to an improved organization of the enzyme in solution.
为了制备并随后表征一种人工双功能酶复合物，将编码半乳糖脱氢酶（荧光假单胞菌）的两个结构基因和荧光素酶的β亚基（哈维弧菌）进行了读码框融合。当该杂交酶与荧光素酶的α亚基一起在大肠杆菌中表达时，表现出半乳糖脱氢酶活性和生物发光。纯化的缀合物用于研究在由双功能酶催化第一个和最后一个反应的顺序三酶反应中可能的邻近效应。中间酶，即心肌黄酶，是单独添加的。与具有单独天然酶的相应参考系统相比，由半乳糖脱氢酶/荧光素酶缀合物组成的工程酶系统在整个酶反应中可显示出高两倍的生物发光。工程酶系统获得的生物发光增加被认为是由于溶液中酶的组织得到了改善。
